Stabilized microbial rennet

ABSTRACT

A stabilized microbial rennet from Mucor miehei is prepared by admixing the solid enzyme product recovered from the fermentation broth with 2-3 percent of fatty acid monoesters of polyoxyethylene sorbitan.

United States Patent 11 1 Schleich 1 1 Oct. 2, 1973 STABILIZED MICROBIAL RENNET [75] Inventor: Hans Schlelch, Staten Island, N.Y.

[73] Assignee: Baxter Lahoratorles, lne., Morton Grove, Ill.

[22] Filed: June 10, 1971 [2]] Appl. No.: 151,992

FOREIGN PATENTS 0R APPLICATIONS 1,201,392 10/1970 Great Britain 962,32l 7/ l 964 Great Britain OTHER PUBLICATIONS Kahn, et al., Influence of Detergents on Enzymic Activity of Cactus Mitochondria, Chemical Abstracts, Vol. 72, 3/1970 (p. 40(63003k)). ODlA5lC.2

Primary Examiner--David M. Nlaft Attorney-Scott J. Meyer 57 ABSTRACT A stabilized microbial rennet from Mucor miehei is prepared by admixing the solid enzyme product recovered from the fermentation broth with 2-3 percent of fatty acid monoesters of polyoxyethylene sorbitan.

4 Claims, No Drawings l STABILIZED MICROBIAL: RENNET bilized milk coagulating enzyme preparation obtained from the growth culture of the microorganism Mucor miehei. v

The conventional method of making cheese involves the use of rennet forcoagulating milk. Rennet is an enzyme preparation generally obtained from thefourth stomach of milk-fed calves. 1n the process ofmakifng cheese, the rennet is added to milk andthe enzyme, rennin, exerts a mildly ,proteolytic action on the casein and other proteins present in milk. This breakdown of the proteins causes the milk to coagulate'and form solid tions. In view of these variable factors, investigators in the field have attempted to findsuitable cheese making substitutes for rennet. I

Recently, the microorganism Mucor miehei has been found to have significant use for the production of a good quality microbial rennet. Such use is described, for example, in British Patent Nos. 1,108,287 and 1,207,892;-U.S. Pat. No. 3,549,390; U.S. Pat. appiications Ser. No. 688,349, filed Dec. 6, l1967,and Ser. No.

95,320, filed Dec. 4, 1970; and Danish Patent Application No. 5118/66. A more complete descriptionofthe microorganism Mucro miehei can be found in the treatise byCooney and Emerson, Thermophilic Fungi," pp. 17-27 (1964), published'by ,H. Freeman and Co., San Francisco and London.

The production of microbial rennetaccording to the aforesaid patents and patent applications generally comprises inoculation of a nutrient medium with a selected culture of Mucor miehei, incubation under aerobic fermentation conditions together with suitable pH, time and temperature conditions, followed by recovery of the resulting rennet from the fermentation broth.

In British Patent No. 1,207,892, a preferred procedure for recovering the rennet product comprises salt or solvent precipitation of the fermentation broth and recovery of a concentrated, fine white powder. While this powder has an acceptable level of stability, it is frequently more desirable to make the rennet available in a liquid form. However, the powdery material does not lend itself to the most desired ease of handling for prep- 1 aration of a liquid product; that is, the powder tends to dust and does not have the optimum solubility. In accordance with the present invention, a stabilized microbial rennet from Mucor miehei is prepared by admixing the solid enzyme product recovered from the fermentation broth with from about 2 percent to about 3 percent by weight of fatty acid mon oesters of polyoxyethylene sorbitan. The esters employed in the invention have from about 12 to about 22 carbon atoms inthe fatty acid moiety and an average of about 20 oxyethylene units per molecule. These esters are commercially available under the trademark Tween and their method of preparation is described ,in detail in U. S. Pat. No. 2,380,166.

A mixture of the stated amount of the ester with the solid enzyme product from the Mucor, miehei fermentationis important. Use of less than about 2 percent of the esterdoes, not provide the desired ease of handling from the standpoint of both dedusting and solubility in aqueous solution, whereas use of greater than about 3 percent of the ester produces a solid porduct that is too wet and lumpy and otherwise is difficult to handle or store.

.The preferred polyoxyethylene sorbitan monoester for admixing with the dry rennet product is polyoxyethylene (20) sorbitan monooleate (Tween 80"). This substance has an optimum balance ofhydrophilic and lipophilicmoieties in the molecule for admixture with the microbial rennet enzyme product in accordance with the objectives of the present invention. The dry rennet product which can be stabilized in accordance with the present invention can comprise the filtered and precipitated culture broth following suitable fermentation as described, for example, in British Patent No. 1,207,892, or it can comprise, for example, similar such products which have been further concentrated or dried such as by vacuum drying, freeze drying,

spray drying and the like drying procedures.

In general, the fermentation is conducted by inoculating a 'medium containing available carbon, nitrogen and trace nutrients with a selected culture of Mucor miehei and fermenting under submerged aerobic conditions at a pH of from about 3 to about 8 and a temperatureof from about 30 C to about C for about 2 to about 14 days. The rennet is preferably recovered pH below about 3 and at a temperatureabove about 40C for at least about minutes.

The preferred strains of Mucor miehel which are used in the invention are available to the public without restriction underthe code designations NRRL A l 3,1 31

. and Al3,0,42 at the Northern Regional Reserach Laboratories, Peoria, 111. 2

lt will be appreciated that the stabilized rennet enzyme preparation of this invention can also contain for their desired effects minor amounts of conventional rennet additives, for example, salts such as sodium chloride, mold inhibitors and preservatives such as sorbic acid, potassium sorbate, or sodium benzoate and other suchnon-toxic, rennetcompatible substances.

when employed in the liquid form, the enzyme preparation preferably is contained in an aqueous base solution containing about 17.5 percent sodium chloride and about 2.5 percent propylene glycol.

Other additiveswhich can be incorporated in the ren- ,net enzyme preparation of the present invention will be apparent to those skilled in the art after reading specification hereof and the appended claims.

The following examples will further illustrate the invention although it will be understood that the invention is not limited to these specific examples.

As used herein, the term SRA means standard rennet activity. The SRA ofa rennet product is defined as the number of rennet units per gram of the product based on a commercially available standard rennet extract known as Chrisl-lansen's Cheese Rennet. This'standard rennet extract, which is a calf rennet, is assigned the value: SRA= 100. Therefore, one rennet unit is the activity of ten milligrams of standard rennet extract. The assay conditions comprise incubation of the rennet product at 25 C in a substrate consisting of percent non-fat dry milk solids in aqueous 0.01 molar calcium chloride.

EXAMPLE 1 Mucor miehei, NRRL A 13,042 is transferred from an agar slant under sterile conditions into a 1 liter Fernbach flask containing 200 ml. of the following aqueous medium:

Soybean flour (Nutrisoy 300 c.) 1.5% Dried Whey 3.0% Degraded Cornstarch 12.0% Water 83.5%

The flask is incubated on a rotary shaker at 37 C for 114 hours. The fermentation broth is filtered and the filtrate shows an activity equal to 125 SRA. The filtrate is then evaporated to about 15120 percent of its vo1- ume. The concentrated evaporate is then adjusted to a pH of 2.6 with 0.1 normal HCl and then heated to 44 C and retained at that temperature for 90 minutesThe pH is readjusted to 4.0, the product is precipitated with methanol, and the solvent is evaporated, yielding a 1ipase-free rennet powder having an activity of 1,000

' SRA. The fine, white powder is than admixed with 2 percent by weight polyoxyethylene (20) sorbitan monooleate. This admixture is storage stable, dust free and readily soluble in aqueous solution. As such, its ease of handling is substantially superior to that of the original powder.

A sample of the dust free solid admixture is readily dissolved in an aqueous solution containing 17.5 percent sodium chloride and 2.5 percent propylene glycol. The rennet activity of this final solution is equal to 1 l5 SRA. Potassium sorbate (1 percent) and sodium benzoate of 1 percent) are added as preservatives.

A cheddar cheese is made by substituting for the rennet preparation ordinarily used in the setting step, an equivalent amount of the liquid mircobial rennet product prepared above. In this procedure, pasteurized whole milk is adjusted to 8688 F and 1 percent by weight of a commercial lacticacid starter solution is added. The microbial rennet is then added to the milk at a rate of 2 to 4 ounces per 1,000 pounds of milk. The mixture is incubated until a curd of satisfactory firmness is obtained. The curd is cut into cubes and then cooked at 100 F for several hours. The curd is separated from the whey and layered into slabs. The milled curd is then saltedwith 3 percent by weight of cheese salt. The solid curd is transferred to hoops, pressed, and then placed in the curing room. The cheese made from the microbial rennet of this example is sampled periodically afterseveral days of curing and is found to possess excellent quality and to be essentially free from offflavors.

EXAMPLE 2 Example 1 is repeated except that the strain of Mucor miehei designated NRRL Al3,13l is substituted for the strain designated A13,042. Similar good quality stability and handling results as in Example 1 are obtained.

EXAMPLE 3 Examples 1 and 2 are repeated except that polyoxyethylene (20) sorbitan monostearate is substituted for the polyoxyethylene (20) sorbitan monooleate employed in said Examples. Similar good quality stability and handling results as in Examples 1 and 2 are obtained.

Various other examples and modifications and adaptations of the foregoing examples can be devised by the person skilled in the art after reading the foregoing specification and the appended claims. All such further examples, modifications and adaptations as come within the true spirit and scope of the invention are included within said appended claims.

l claim:

1. A stabilized microbial rennet enzyme preparation comprising the solid enzyme product recovered from the fermentation broth of Mucor miehei admixed with from about 2 percent to about 3 percent by weight of fatty acid monoesters of polyoxyethylene sorbitan having from about 12 to about 22 carbon atoms in the fatty acid moiety and an average of about 20 oxyethylene units per molecule.

2. The'microbial rennet enzyme preparation of claim 1 in dilute aqueous solution containing additionally about 17.5 percent sodium chloride and about 2.5 percent propylene glycol.

3. The microbial rennet enzyme preparation of claim 1 in which the Mucor miehei is selected from the group consisting of the strains designated NRRL A 1 3,131 and 13,042.

4. The microbial rennet enzyme preparation of claim 2 in which the Mucor miehei is selected from the group consisting of the strains designated NRRL A l 3,1 31 and 

2. The microbial rennet enzyme preparation of claim 1 in dilute aqueous solution containing additionally about 17.5 percent sodium chloride and about 2.5 percent propylene glycol.
 3. The microbial rennet enzyme preparation of claim 1 in which the Mucor miehei is selected from the group consisting of the strains designated NRRL A13,131 and 13,042.
 4. The microbial rennet enzyme preparation of claim 2 in which the Mucor miehei is selected from the group consisting of the strains designated NRRL A13,131 and 13,042. 